Method for immobilisation of (an) affinity reagent(s) on a hydrophobic solid phase

ABSTRACT

The invention relates to a method of immobilizing an affinity reagent or affinity reagents on a hydrophobic solid phase which can be used in biological assays for analyte detection. The invention relates more particularly to a method of immobilizing an affinity reagent on a hydrophobic solid phase functionalized by a carboxyl group, said method comprising a step for activation of said solid phase and a step for coupling of the affinity reagent to said solid phase, characterized in that the step for activation of said solid phase uses a combination of a carbodiimide and a phosphate buffer in the presence of a co-activator and in an acid medium, and in that the coupling step is performed in a basic medium.  
     The invention further relates to the reactive complexes obtained by this method and to their use in immunoassay kits, hybridization kits or enzymatic assay kits.

[0001] The invention relates to a method for immobilisation of (an)affinity reagent(s) on a hydrophobic solid phase which can be used inbiological assays for analyte detection, to the reactive complexesobtained by this method and to the use of said complexes in biologicalassay kits.

[0002] Biological analysis assays which use reagents with a mutualaffinity have been known for decades. Said reagents will hereafter bereferred to by the term ‘affinity reagents’ or ‘affinity pair reagents’.Thus it is known how to search for and detect one member of an affinitypair by means of the other. Biological assays which utilize an affinityreaction between affinity reagents, or ‘affinity assays’, includeenzymatic analyses utilizing e.g. an enzyme and its substrate,qualitative or quantitative immunoassays initiated by the pioneeringwork of Berson and Yalow (1959) and involving the reaction of anantibody with the corresponding antigen or hapten, more recently nucleicacid hybridization assays utilizing a target oligo-nucleotide orpoly-nucleotide with a complementary nucleotide probe capable ofhybridizing specifically therewith, etc.

[0003] Since the 1960s attempts have been made to use reactive solidphases (or solid supports) in affinity assays requiring a separation ofthe free complexes and the bound complexes obtained, in order tosimplify this separation step.

[0004] Affinity reagents can be immobilized on a solid phase by covalentcoupling, for example with the aid of glutaraldehyde. It is also known,since the work of Catt and Tregear in 1967, to immobilize an affinityreagent on a solid phase by simple passive adsorption.

[0005] Immobilization by passive adsorption has the advantage ofsimplicity, but it can cause inappropriately immobilized reagent to bereleased into a liquid medium.

[0006] Solid-phase covalent couplings generally have the advantage ofproducing the final reagent with a greater stability, but they alsoenable more affinity reagent to be immobilized on a solid support. Thereare a large number of solid-phase covalent coupling modes and agentscurrently available: non-limiting examples which may be mentioned arecouplings with glutaraldehyde, cyanogen bromide and carbodiimides, inthe presence or absence of a co-activator such as DMAP(dimethylaminopyridine), HOBt (1-hydroxybenzotriazole),N-hydrosuccinimide or s-NHS (sulfo-N-hydroxysuccinimide), which are wellknown to those skilled in the art.

[0007] The different protocols for immobilizing an affinity reagent on asolid phase by covalent coupling are carried out in one, two or eventhree steps. In one-step couplings, all the ingredients are brought intocontact with one another. Two-step couplings generally involve a firststep in which the solid support is activated by a so-called ‘activator’,then washed to remove any excess unreacted activator, and finallybrought into contact with the affinity reagent, enabling the actualcoupling to be carried out in a second step.

[0008] Numerous solid phases or supports are known and used: hydrophilicsolid phases (for example Sephadex® marketed by Pharmacia) andhydrophobic solid phases (for example polypropylene, polystyrene,latices, etc.). The latter are generally rendered reactive viafunctional groups grafted on beforehand, such as amine, carboxyl, tosyl,aldehyde, hydroxyl, thiol, chloromethyl, hydrazide and other groups.

[0009] Different techniques have been proposed for the covalent couplingof affinity reagents to hydrophobic solid phases functionalized by acarboxyl group: various combinations of buffer and activator are usedsuch as the combination of MES (2-[N-morpholino]ethanesulfonic acid)with a carbodiimide. An example which may be mentioned is the covalentcoupling of antibodies to carboxylic latices in the presence of MES andEDC (1-ethyl-3-[3-dimethylamino-propyl]carbodiimide), described by D.Bastos-Gonzalez et al., J. of Colloid and Interface Science, 176,232-239 (1995), or by C. Bieniarz et al., Bioconjugate Chem., 1996, 7,88-95.

[0010] However, it has long been known that the combination of phosphatebuffer and carbodiimide—as activator—is to be avoided becausecarbodiimides have the disadvantage of activating phosphates as well andhence of losing a large part of their reactivity. This resultssimultaneously in an insufficient efficacy of covalent coupling, a highproportion of passive adsorption and, as a consequence, an instabilityof the products obtained (Wong, S. in ‘Chemistry of protein conjugationand crosslinking’, chapter 6, page 196 (1991), and M. A. Gilles et al.,Analytical Biochemistry, 184, 244-248 (1990)).

[0011] Bangs (Bangs Laboratories Inc., Tech. Note #13c, Covalentcoupling protocols, page 3) has proposed a protocol for the activationof hydrophobic solid phases functionalized by a carboxyl group with theaid of the carbodiimide EDC in aqueous solution, but the resultsobtained are far from satisfactory from the point of view of couplingefficacy. In fact, the coupling is not quantitative and the proportionof passive adsorption is high compared with the covalent coupling (cf.Example 1 below).

[0012] In general, with all the techniques of the prior art aimed atusing covalent coupling to immobilize an affinity reagent on ahydrophobic solid phase functionalized by a carboxyl group, it isobserved that the actual covalent coupling between the affinity reagentand the solid phase is simultaneously accompanied by the unwantedco-existence of a substantial degree of fixing of said affinity reagentto said solid phase by passive adsorption. In other words, thesetechniques have the disadvantage of being incapable of allowing thedesired maximum degree of covalent fixing. As passive adsorption cannotbe controlled in favor of covalent coupling, it follows that thesetechniques do not make it possible to optimize the covalent fixing ofthe affinity reagent or hence to obtain reproducible covalent couplings.Consequently they give rise to products which are unstable over time.

[0013] There is therefore a genuine need for a method of immobilizing anaffinity reagent on a hydrophobic solid phase functionalized by acarboxyl group which makes it possible reproducibly to control andoptimize the covalent character of the coupling while at the same timeminimizing the possibility of simultaneous passive adsorption.

[0014] It has now been found, surprisingly, that it is possiblereproducibly to control and optimize the covalent character of areaction for immobilizing an affinity reagent on a hydrophobic solidphase functionalized by a carboxyl group with the aid of a combinationof a carbodiimide and a phosphate buffer as activator.

[0015] The invention therefore relates to a method of immobilizing anaffinity reagent on a hydrophobic solid phase functionalized by acarboxyl group, said method comprising a step for activation of saidsolid phase and a step for coupling of the affinity reagent to the solidphase, and being characterized in that the step for activation of saidsolid phase uses a combination of a carbodiimide and a phosphate bufferin an acid medium, in the presence of a co-activator, and in that thestep for coupling of the affinity reagent is performed in a basicmedium.

[0016] Any carbodiimide used in this field as a carboxyl groupactivator, and in the field of peptide synthesis, can be used for thepurposes of the invention.

[0017] Examples of carbodiimides are described especially by Lundblad,R. L. et al., Chemical Reagents for Protein Modification, vol. 2, chap.4, CRC Press; Boca Raton, F. L. and Marion Mikolajczyk et al.,Tetrahedron, vol. 37, pp. 233-284 (1981).

[0018] CMC (N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimidemethyl-p-toluenesulfonate) and EDC(1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide) may be mentioned inparticular among the carbodiimides which can be used as activatorswithin the framework of the invention, CMC being particularly preferred.

[0019] The carbodiimide must be used in excess relative to the COOHgroup. The amount used is advantageously 20 to 50 molar equivalents perCOOH group.

[0020] ‘Phosphate buffer’ is understood within the framework of theinvention as meaning any conventional phosphate buffer (sodium and/orpotassium) used at a concentration generally ranging from 30 to 200 mM,50 mM phosphate buffer being more particularly preferred.

[0021] It is possible to use any co-activator employed in this field.Examples of co-activators are described especially by Staros, J. V.,Biochemistry, 21, 3950-3955 (1982); O'Sullivan, M. J. et al., Anal.Biochem., 100, 100-108 (1979); Abdella, P. M. et al., Biochem. Biophys.Res. Commun., 87, 734-742 (1979).

[0022] s-NHS (sulfo-N-hydroxysuccinimide), HOBt (1-hydroxybenzotriazole)and N-hydroxysuccinimide can be employed in particular among theco-activators which can be used according to the invention, s-NHS(sulfo-N-hydroxysuccinimide) being particularly preferred.

[0023] Like the carbodiimide, the co-activator must be used in excessrelative to the COOH group. The amount used is advantageously 3 to 10molar equivalents per COOH group.

[0024] ‘Acid medium’ in the activation step of the method of theinvention is understood as meaning any medium with a pH ranging fromabout 4 to about 6.5, a pH of 6 being more particularly preferred, theacid character of said medium being conferred by the phosphate buffer.

[0025] ‘Basic medium’ in the coupling step of the method of theinvention is understood as meaning a medium with a pH ranging from about7.2 to about 10.5, a medium containing 50% of a buffer of pH 8.5 beingmore particularly preferred.

[0026] The basic character of the medium is obtained by usingappropriate conventional buffers, for example a borate buffer or aphosphate buffer/borate buffer mixture.

[0027] Thus, in a preferred embodiment of the invention, the step foractivation of the solid phase uses a combination of 20 to 50 molarequivalents per COOH group of CMC, in 30-200 mM phosphate buffer, in thepresence of 3 to 10 molar equivalents per COOH group ofsulfo-N-hydroxysuccinimide co-activator, in an acid medium with a pHranging from about 4 to about 6.5, and the coupling is carried out in abasic medium with a pH ranging from about 7.2 to about 10.5.

[0028] In a particularly preferred embodiment of the invention, the stepfor activation of the solid phase uses a combination of 30 molarequivalents per COOH group of CMC, in 50 mM KH₂PO₄ phosphate buffer, inthe presence of 5 molar equivalents per COOH group ofsulfo-N-hydroxysuccinimide co-activator, at pH 6, and the coupling iscarried out in a medium containing 50% of a borate buffer of pH 8.5.

[0029] The judicious combination of activation parameters—carbodiimide,phosphate buffer, co-activator and acid pH—associated with the couplingconditions described above is essential for implementation of the methodof the invention.

[0030] The affinity reagents which can be immobilized within theframework of the invention are any compounds which have an amine groupor can be artificially provided with an amine group. Such affinityreagents which may be mentioned are proteins, peptides, immunoglobulins,antigens, haptens, antibodies, enzymes, enzyme substrates,oligonucleotides, polynucleotides, etc., as well as any other biologicalreagent known to those skilled in the art.

[0031] ‘Hydrophobic solid phase’ is understood in the presentdescription as meaning solid phases consisting of hydrophobic polymerscommonly used in this field, for example polypropylenes andvinylaromatic polymers such as polystyrenes, and especially the laticesof these polymers. These solid phases are functionalized by a carboxylgroup using the techniques well known to those skilled in the art. Inthis connection, reference may be made e.g. to the article by OttewillR. H. et al. in Kolloid Zu Z. Polymere, 215, 161-166 (1967).

[0032] Among the hydrophobic solid phases functionalized by a carboxylgroup which can be used according to the invention, there may bementioned latex particles, for example those known under the markEstapor® (Prolabo, France), Dynabeads® magnetic particles from Dynal,Polybead® microspheres from Polysciences, Inc., and equivalents thereof,etc.

[0033] The invention further relates to the reactive solid complexesobtainable by the method according to the invention, such as solidphase/antigen complexes, solid phase/hapten complexes, solidphase/antibody complexes, etc., which can be used in immunoassays, solidphase/oligonucleotide or polynucleotide complexes, which can be used innucleic acid hybridization assays, amplification assays, etc., solidphase/enzyme complexes or solid phase/enzyme substrate complexes, etc.

[0034] The invention further relates to the use of these complexes inkits for biological assays, non-restricting examples being immunoassays,nucleic acid hybridization assays, nucleic acid amplification assays,enzymatic assays, etc., known to those skilled in the art, whetherqualitative or quantitative.

[0035] The invention will be understood more clearly with the aid of thefollowing Examples, which are given simply by way of illustration andmust not in any way be understood as restricting the scope of theinvention.

EXAMPLE 1

[0036] Coupling of a Peptide to Carboxylic Beads

[0037] a) Reagents

[0038] a1) Carboxylic Beads

[0039] Magnetic carboxylic latex beads produced by Prolabo, France(reference Estapor M1-070/60) were used as the hydrophobic solid phasecarrying a carboxyl group. They consist of polydisperse particles ofpolystyrene and iron oxide, functionalized by COOH groups. The solidphase used has the following characteristics: mean diameter (0.8 82 m),percentage of iron (62%), degree of flnctionalization (150 μeq COOH/g).It takes the form of a 0.1 g/ml aqueous suspension.

[0040] All the bead washing steps are performed as follows:

[0041] In each experiment the magnetic beads present in the test tubesare separated from the solutions with a magnetized support. Thesupernatants are removed with a pipette, the magnetic beads being heldin said tubes by the magnetized support. After each addition of a newsolution or new buffer, the beads are resuspended by vortexing for about10 seconds.

[0042] A wash comprises the addition of the washing solution, theresuspension of the beads and the removal of this solution bymagnetization.

[0043] a2) Peptide

[0044] The peptide of 17 amino acids having the following sequence wasused in this Example: KGSYSVDHFRWGRPVSG-NH2.

[0045] This peptide was prepared by the procedure described by E.Atherton and R. L. Sheppard in ‘Solid phase peptide synthesis, apractical approach’, IRL PRESS (1989), Oxford University Press, pp.25-34.

[0046] The phosphate buffer solution used was a 50 mM aqueous solutionof KH₂PO₄ of pH 6.

[0047] All the operations were carried out at room temperature, i.e. at19-24° C.

[0048] b) Immobilization of the Peptide on the Carboxylic Beads

[0049] b1) Covalent Coupling of the Peptide to the Carboxylic Beads

[0050] Step 1: Washing of the Carboxylic Beads:

[0051] 50 μl of magnetic carboxylic latex beads in a test tube arewashed twice with 750 μl of 0.1 ImM NaOH of pH 9, once with 750 μl ofdouble-distilled water and finally once with 750 μl of phosphate buffer.

[0052] Step 2: Activation of the Carboxylic Beads:

[0053] 250 μl of phosphate buffer, 200 μl (30 eq/COOH group) of anaqueous solution of CMC [N-cyclohexyl-N′-(2-morpholino)carbodiimidemethyl-p-toluene-sulfonate (Fluka)] and 50 μl (5 eq/COOH) of an aqueoussolution of [s-NHS (sulfo-N-hydroxysuccinimide (Pierce)] are added tothe residue of washed beads obtained in step 1.

[0054] The reaction mixture is incubated for 1 hour at room temperature,with shaking. The beads are then washed with 500 μl of phosphate buffer.

[0055] Step 3: Coupling of the Peptide to the Beads:

[0056] 250 μl of phosphate buffer and 250 μl of a 37.5 mM borate/50 mMNaCl buffer solution of pH 8.5, containing 0.25 eq (based on the COOHgroups) of the peptide described in section a2), are added to theresidue of activated beads obtained in step 2.

[0057] The reaction mixture is incubated for 1 hour at room temperature,with shaking. The beads are separated by magnetization and thesupernatant is retained for determination by HPLC (cf. section c)below). After washing, the beads are kept in PBS of pH 7.4 or any othersuitable equivalent buffer.

[0058] b2) Passive Adsorption of the Peptide on the Carboxylic Beads

[0059] A coupling of the ‘passive’ type—also called ‘passiveadsorption’—is performed using exactly the same reagents as above exceptthat the activator and coupling agent, CMC and s-NHS, are omitted. Aftercoupling of the ‘passive’ type, the beads are separated by magnetizationand the supernatant is retained for determination by HPLC (cf. sectionc)).

[0060] c) Calculation of the Covalent Coupling Yield and Evaluation ofthe Passive Coupling by HPLC

[0061] The couplings are followed by reversed-phase high performanceliquid chromatography (HPLC) on an apparatus (e.g. Waters) with anapolar stationary phase (C18) and a polar mobile phase (gradient:acetonitrile/0.08% aqueous TFA—0.1% TFA).

[0062] At the end of each coupling (covalent or passive), 30 μl of thesupernatant obtained in section b1) or b2) are injected into the HPLCapparatus.

[0063] An identical volume of ‘control solution’ (i.e. the same peptidesolution (in 250 μl of 37.5 mM borate/50 mM NaCl buffer of pH 8.5 and250 μl of phosphate buffer) as that used in the couplings except that itis not coupled to the magnetic beads) is also injected into the HPLCapparatus.

[0064] The HPLC chromatograms, obtained by measuring the absorbance at214 nm as a function of the time T expressed in minutes, are shown inFIGS. 1 to 3. The chromatogram of FIG. 1 shows the peak obtained with acontrol solution (assay without coupling to the magnetic beads) at T₀,said solution serving as a reference by indicating the total amount ofpeptide introduced.

[0065] The chromatogram of FIG. 2 shows the peak obtained with asupernatant taken at T_(0+1 hour) at the end of the covalent couplingdescribed in section b1).

[0066] The chromatogram of FIG. 3 shows the peak obtained with asupernatant taken at T_(0+1 hour) at the end of the passive adsorptiondescribed in section b2).

[0067] For each chromatogram the peak area is integrated by a softwareprogram (e.g. Millenium software). This measures the area A0 for thecontrol solution, the area Al for the covalent coupling and the area A2for the ‘passive type’ coupling.

[0068] The coupling yield is thus evaluated by a back determination,known to those skilled in the art, using the following formula:${\% \quad {covalent}\quad {coupling}} = {\frac{\left( {{A\quad 0} - {A\quad 1}} \right)}{A\quad 0} \times 100}$${\% \quad {‘{passive}’}\quad {coupling}} = {\frac{\left( {{A\quad 0} - {A\quad 2}} \right)}{A\quad 0} \times 100}$

[0069] d) Comparison of the Coupling According to the Invention withCouplings According to the Prior Art

[0070] The study was carried out on 3 protocols of the prior art and theprotocol according to the invention. A coupling under ‘passive’conditions, omitting the coupling agent and activator, was performed inparallel for all the protocols. The yields of covalent coupling andpassive coupling were evaluated by the method described in c).

[0071] Protocols of the Prior Art:

[0072] ‘Bangs’ protocol, Bangs Laboratories Inc., Tech. note #13c,Covalent coupling protocols, page 3.

[0073] The beads were washed in a preactivation buffer (50 mM phosphatebuffer of pH 4.5) and then activated with an aqueous solution of EDC inthe presence of a small percentage (20%) of preactivation buffer. Thecoupling was performed in 0.2 M borate buffer of pH 8.5. After coupling,the unreacted carboxyl groups were blocked with ethanolamine solution.The beads were kept in a buffer containing BSA, glycine, Tween®detergent and sodium azide.

[0074] J. Sackrison protocol, Covalent coupling to latex particles anddiagnostic development using microspheres, the latex course, 1997.

[0075] The beads were washed several times with a 10 mM borate buffersolution of pH 8.5, with a 10 mM sodium acetate solution of pH 5.0 andwith a 50 mM diethanolamine solution of pH 10.2 and then activated witha solution of CMC in 50 mM diethanolamine buffer of pH 10.2. Thecoupling was performed in 100 mM phosphate/150 mM NaCl buffer of pH 7.4.

[0076] protocol 1 described by D. Bastos-Gonzalez et al., J. of Colloidand Interface Science, 176, 232-239, 1995.

[0077] Latex beads were added to a buffer solution (MES) of pH 5.6. Thecoupling was performed in a weakly acidic medium (in MES buffer of pH5.6). An aqueous solution of EDC was added and the sample was thenincubated at room temperature. After coupling, the excess carboxylgroups were blocked by treatment with ethanolamine.

[0078] The results obtained by HPLC determination are indicated in TableI in the form of the coupling yields (%) and the difference betweencovalent coupling and passive adsorption (Δ): TABLE I % of % ofpassively covalently coupled coupled Δ Protocol peptide peptide(covalent − passive) Bangs 47 57 10 Latex course 80 82 2 (J. Sackrison)Protocol 1 8 13 5 (D. Bastos-Gonzalez et al.) Protocol according to the54 100 46 invention

[0079] The best performance characteristics are obtained with thecoupling protocol according to the invention when compared with thoseobtained with the protocols of the prior art; thus, with the couplingprotocol according to the invention, the covalent coupling yield isquantitative (100%) and the difference between covalent coupling andpassive coupling is appreciable, in contrast to the protocols of theprior art, where the difference between the 2 forms of coupling isrelatively insignificant.

[0080] e) Reproducibility of the Coupling Method According to theInvention

[0081] The reproducibility of the coupling method according to theinvention was studied on the one hand by performing 3 couplings on oneand the same batch of beads, and on the other hand by performing acoupling on a different batch of beads.

[0082] A covalent coupling according to the protocol described insection b1) (above) and a coupling of the ‘passive’ type according tothe protocol described in section b2) (above) were performed in parallelin each experiment. Calculation of the coupling yield and evaluation ofthe passive coupling were determined according to protocol c) above.

[0083] The results obtained by HPLC determination are indicated in TableII in the form of the coupling yields (%) and the difference betweencovalent coupling and passive adsorption (Δ): TABLE II Batch of % ofpassively % of covalently Δ beads coupled peptide coupled peptide(covalent − passive) 477 52 98 46 477 54 100 46 477 58 100 42 583 56 9337

[0084] These results show that the reproducibility is excellent with oneand the same batch (batch 477). With a different batch (batch 583) thecoupling yield is totally acceptable and comparable to the yieldobtained with the first batch.

EXAMPLE 2

[0085] a) Coupling of Bovine Serum Albumin (BSA) to Magnetic CarboxylicBeads

[0086] 175 μg of BSA (Pantex) are coupled under the conditions describedin section b1) of Example 1. The actual coupling reaction is carried outfor 22 hours (instead of 1 hour) at room temperature.

[0087] A passive adsorption is performed under the same conditionsexcept that the coupling agent, CMC, and the co-activator, s-NHS, areomitted. After coupling, the beads are separated by magnetization andthe supernatant is retained for determination by HPLC.

[0088] b) Comparison of the BSA Coupling According to the Invention withCouplings of the Prior Art

[0089] The protocol according to the invention was compared with 2protocols of the prior art, namely the ‘Bangs’ protocol and the ‘latexcourse’ protocol (J. Sackrison).

[0090] The covalent coupling and passive adsorption yields using the 3protocols were evaluated by the method described in section c) ofExample 1.

[0091] The results obtained by HPLC determination are indicated in TableIII in the form of the coupling yields (%) and the difference betweencovalent coupling and passive adsorption (Δ): TABLE III % of passively %of covalently Δ Protocol coupled BSA coupled BSA (covalent − passive)Bangs 13 17 4 Latex course 31 34 3 (J. Sackrison) Protocol 25 65 40according to the invention

[0092] The results show the superiority of the protocol of the inventioncompared with the protocols of the prior art since the former affords amuch higher coupling Δ (covalent—passive).

EXAMPLE 3

[0093] Application to a Diagnostic Test for the Detection of Anti-HIVAntibodies

[0094] As indicated below, the beads obtained by the method of theinvention were used to detect anti-HIV antibodies in a known ELISA test,namely the Access® HIV 1-2 New test marketed by and available fromBio-Rad Laboratories, Marnes la Coquette, France, catalog number 34 020,in which an anti-HIV-2-specific antibody is sandwiched between a captureantigen immobilized on magnetic beads and an antigen labeled with anenzyme. Disclosure and measurement of the signal are effected by addinga chemoluminescent enzyme substrate and reading off the luminescencegenerated.

[0095] a) Materials and Methods

[0096] a1) Capture Antigen (Peptide)

[0097] A peptide of 27 AA containing the essential immunodominantepitope, namely the heptapeptide CAFRQVC of gp36 of HIV-2, wassynthesized by the above-mentioned method of E. Atherton and R. L.Sheffard and then coupled to BSA by covalent coupling with the aid ofthe homobifunctional reagent bis-(sulfosuccinimyl) suberate. TheBSA/HIV-2 peptide conjugate obtained is hereafter referred to as‘BSA/HIV-2’.

[0098] a2) Immobilization of the Capture Antigen

[0099] The above BSA/HIV-2 conjugate was then coupled to magneticcarboxylic latex beads (Estapor) according to the covalent couplingprotocol of the invention and according to a protocol of the prior art.

[0100] 12 μg of BSA/HIV-2 conjugate were coupled to 100 μl of beads bythe method described in Example 1b).

[0101] 12 μg of BSA/HIV-2 conjugate were coupled to 100 μl of beads by amethod of the prior art (protocol 1 of Bastos-Gonzalez: cf. Example1d)).

[0102] The resulting magnetic beads carrying BSA/HIV-2 peptide arehereafter referred to as ‘BSA/HIV-2 beads’.

[0103] a3) Disclosure Antigen

[0104] The same peptide of 27 AA containing the essential immunodominantepitope, namely the heptapeptide CAFRQVC of gp36 of HIV-2, as that usedin a1) above was coupled to alkaline phosphatase (hereafter referred toas ‘ALP’), from Biozyme, by covalent coupling with the aid of thehomobifunctional reagent bis(sulfosuccinimyl) suberate. The HIV-2peptide/ALP conjugate obtained is hereafter referred to as‘peptide/ALP’.

[0105] a4) Detection of the Signal

[0106] Disclosure is obtained using a dioxetane-based substrate specificfor alkaline phosphatase. The signal is read off on an Access®luminometer available from Bio-Rad Laboratories, France. The signal isexpressed in RLU (Relative Luminescence Units).

[0107] a5) Samples

[0108] The assay was performed using human sera positive in anti-HIV-2antibodies, diluted in negative human serum (qc1, qc2, qc3), and a serumnegative in anti-HIV antibodies (CO).

[0109] b) Assay Protocol

[0110] Two series were carried out in parallel.

[0111] b1) 50 μl of serum were brought into contact with 50 μg ofBSA/HIV-2 beads obtained according to the covalent coupling protocol ofthe invention, and with 350 μl of peptide/ALP conjugate. The mixture wasincubated for 20 minutes at 37° C., the beads were then separated bymagnetization and the supernatant was removed.

[0112] 200 μl of substrate were added and incubation was carried out for5 minutes at 37° C.

[0113] The reading was taken and the RLU recorded. The results areexpressed as the ‘Signal/CO’ RLU ratio: cf. Table IV.

[0114] b2) 50 μl of serum were brought into contact with 50 μg ofBSA/HIV-2 beads obtained according to protocol 1 of Bastos-Gonzalez, andwith 350 μl of peptide/ALP conjugate.

[0115] The remainder of the protocol is identical to that of procedureb1) above.

[0116] c) Results and Comparison with the Prior Art

[0117] The results obtained with the coupling according to the inventionand those obtained with the coupling according to protocol 1 areindicated in Table IV: TABLE IV Protocol according Protocol 1 to theinvention Serum tested RLU SIGNAL/C0 RLU SIGNAL/C0 C0 15,205 0.98 10,7671.00 15,741 1.02 10,771 1.00 mean 15,473 10,769 qc1 171,048 11.051,894,150 175.89 168,968 10.92 1,904,600 176.86 qc2 15,517 1.00 181,88816.89 15,444 1.00 183,826 17.07 qc3 12,731 0.82 80,437 7.47 12,591 0.8180,462 7.47

[0118] The results show that, for the positive samples, the ‘Signal/C0’ratio obtained by the protocol according to the invention isconsiderably greater than that obtained by protocol 1 of the prior art.This translates into a much better immuno-reactivity and a betteranalytical sensitivity and very clearly reflects the fact that thecoupling method according to the invention has afforded an optimumcovalent coupling compared with protocol 1.

EXAMPLE 4

[0119] Stability Tests on Magnetic Carboxylic Latex Beads Coated withBSA/HIV-2 Conjugate (‘BSA/HIV-2 beads’) of the Invention

[0120] A long-term stability study—7 months, 12 months and 18 months at+4° C.—was carried out using the protocol of Example 3 for BSA/HIV-2beads (batch C7P184A obtained in Example 3 from beads of initial batch477, cf. above). Table V summarizes the results obtained, which showthat the batch did not significantly lose immunoreactivity in 18 monthsat +4° C. In the results in Table V below, the expression ‘SIGNAL/C0’has been replaced by the abbreviated form ‘S/C0’ for the sake ofpresentation. TABLE V Serum T0 T = 7 months T = 12 months T = 18 monthstested RLU S/C0 RLU S/C0 RLU S/C0 RLU S/C0 C0 12,144 13,962 12,89812,768 11,866 13,692 13,855 12,773 13,586 mean 12,005 1.00 13,827 1.0013,446 1.00 12,771 1.00 qc2 139,029 184,633 170,026 182,311 141,796185,985 173,015 182,952 173,033 mean 140,413 12 185,309 13 172,025 13182,632 14 qc1 1,732,230 2,166,040 2,159,050 2,076,950 1,799,8802,142,940 2,279,010 2,127,930 2,102,220 mean 1,766,055 147 2,154,490 1562,180,093 162 2,102,440 165

[0121] These results clearly show that the method according to theinvention affords complexes of affinity reagents (immobilized on ahydrophobic solid phase functionalized by a carboxyl group) which have ahigh stability over time.

EXAMPLE 5

[0122] Covalent Coupling of Nucleic Acid to Magnetic Carboxylic LatexBeads

[0123] a) Materials and Methods

[0124] 5×SSC and 2×SSC buffers are prepared by the method described inManiatis T. et al., Molecular Cloning. A laboratory manual, Cold SpringHarbor laboratory, New York (1982).

[0125] Probes (DNA) functionalized in the 5′ position by an amine groupare obtained via an automatic synthesizer using the commercial reagentaminolink 2 from Perkin Elmer, and are referred to below as 5′-NH₂probes.

[0126] The analyte used is either RNA or DNA. The performancecharacteristics of the probe coupling are evaluated in a sandwichhybridization format on an Access® apparatus from Beckman.

[0127] An analyte-specific DNA probe is coupled to magnetic particlesaccording to the protocol described in b) below. It serves to capturethe analyte (capture probe).

[0128] Disclosure is effected with the aid of an analyte-specificdetection probe (different from the first probe) labeled, e.g. with theenzyme alkaline phosphatase, by methods known to those skilled in theart.

[0129] Examples of the non-radioactive labeling of probes are describede.g. in patent FR 78.10975, by M. S. Urdea et al., Nucleic Acids Symp.Ser., 24, 1991, 197-200, or by R. Sanchez-Pescador, J. Clin. Microbiol.,26, 1988, 1934-1938.

[0130] The yield of the covalent coupling of nucleic acid to magneticcarboxylic latex beads is evaluated as in Example 1 by HPLC underconditions adapted to nucleic acids: for example stationary phase (C18)and mobile phase (gradient of buffer A: 10⁻² M triethylammonium acetate,and buffer B: acetonitrile/A: 95/5).

[0131] b) Covalent Coupling of a 5′-NH₂ Probe to Magnetic CarboxylicLatex Beads

[0132] 20 μg of a 5′-NH₂ probe (i.e. a probe carrying an NH₂ group inthe 5′ position) are coupled to 200 μl of Estapor M1-070/60 magneticcarboxylic latex beads under similar conditions to those of Example b1).

[0133] The beads are then washed twice with 500 μl of 5×SSC buffer andtwice with 500 μl of 2×SSCC buffer. They are kept in 2×SSC buffercontaining 0.02% of NaN₃.

[0134] Hybridization assay: Hybridization of the capture and detectionprobes with the analyte can be effected separately (in two steps) orsimultaneously (in one step), especially by one of the methods describedby Langhale and Malcolm, Gene, 36, 1985, 201-210, by Ranki et al., Gene,21, 1993, 77-85, by Dunn and Hassel, Cell, 12, 1977, 23-36, or by Rankiand Soderlund in patents U.S. Pat. No. 4,486,539 and U.S. Pat. No.4,563,419.

[0135] Those skilled in the art will be capable of reproducinghybridization experiments without difficulty and comparing the couplingmethod according to the invention with coupling techniques of the priorart. Coupling techniques of the prior art which may be mentioned are thefixing of the capture probe to the solid support by well-known methods,especially by passive adsorption or covalent coupling (Cook et al.,Nucleic Acids Res., 16, 1988, 4077-4095; Nagata et al., FEBS Lett., 183,1985, 379-382; M. Longlaru et al., EP 420 260 A2; T. Gingeras et al., EP276 302; E. Kornes and L. M. Kornes, EP 446 260).

1 2 1 17 PRT Artificial Sequence Peptide 1 Lys Gly Ser Tyr Ser Val AspHis Phe Arg Trp Gly Arg Pro Val Ser 1 5 10 15 Gly 2 7 PRT Humanimmunodeficiency virus type 2 PEPTIDE (1)..(7) immunodominant epitope ofgp36 2 Cys Ala Phe Arg Gln Val Cys 1 5

1. Method of immobilizing an affinity reagent on a hydrophobic solidphase functionalized by a carboxyl group, said method comprising a stepfor activation of said solid phase and a step for coupling of theaffinity reagent to said solid phase, characterized in that the step foractivation of said solid phase uses a combination of a carbodiimide anda phosphate buffer in the presence of a co-activator and in an acidmedium with a pH ranging from about 4 to about 6.5, and in that thecoupling step is performed in a basic medium with a pH ranging fromabout 7.2 to about 10.5.
 2. Method according to claim 1, characterizedin that the carbodiimide used is a compound selected from the groupcomprising CMC (N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimidemethyl-p-toluenesulfonate) and EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide).
 3. Method according toclaim 1 or 2, characterized in that the co-activator used is a compoundselected from the group comprising s-NHS (sulfo-N-hydroxy-succinimide),HOBt (1-hydroxybenzotriazole) and N-hydroxysuccinimide.
 4. Methodaccording to any one of claims 1 to 3, characterized in that thecarbodiimide used is CMC(N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimidemethyl-p-toluenesulfonate) and the co-activator used is s-NHS(sulfo-N-hydroxy-succinimide).
 5. Method according to any one of claims1 to 4, characterized in that the carbodiimide is used in an amount of20 to 50 molar equivalents per COOH group.
 6. Method according to anyone of claims 1 to 4, characterized in that the co-activator is used inan amount of 3 to 10 molar equivalents per COOH group.
 7. Methodaccording to any one of claims 1 to 6, characterized in that the stepfor activation of the solid phase uses a combination of 20 to 50 molarequivalents per COOH group of CMC, in 30-200 mM phosphate buffer, in thepresence of 3 to 10 molar equivalents per COOH group ofsulfo-N-hydroxysuccinimide co-activator, in an acid medium with a pHranging from about 4 to about 6.5, and in that the coupling is performedin a basic medium with a pH ranging from about 7.2 to about 10.5. 8.Method according to any one of claims 1 to 6, characterized in that thestep for activation of the solid phase uses a combination of 30 molarequivalents per COOH group of CMC, in 50 mM KH₂PO₄ phosphate buffer, inthe presence of 5 molar equivalents per COOH group ofsulfo-N-hydroxysuccinimide co-activator, at pH 6, and the coupling isperformed in a medium containing one volume of a buffer of pH 8.5 andone volume of said phosphate buffer.
 9. Method according to any one ofclaims 1 to 8, characterized in that the affinity reagent contains anamine group or can be artificially provided with an amine group. 10.Method according to claim 9, characterized in that the affinity reagentis selected from the group comprising proteins, peptides,immunoglobulins, antigens, haptens, antibodies, enzymes, enzymesubstrates, oligonucleotides and poly-nucleotides.
 11. Reactive solidcomplex obtained by a method according to any one of claims 1 to 10,characterized in that it is selected from the group comprising solidphase/antigen complexes, solid phase/protein complexes, solidphase/peptide complexes, solid phase/hapten complexes, solidphase/antibody complexes, solid phase/immunoglobulin complexes, solidphase/oligonucleotide complexes, solid phase/polynucleotide complexes,solid phase/enzyme complexes and solid phase/enzyme substrate complexes.12. Use of the complex according to claim 11 in immunoassay kits,hybridization kits and enzymatic assay kits.